Cellular growth and differentiation appear to be initiated, promoted, maintained and regulated by a multiplicity of stimulatory, inhibitory and synergistic factors and hormones. The alteration and/or breakdown of the cellular homeostasis mechanism seems to be a fundamental cause of growth related diseases, including neoplasia. Growth modulatory factors are implicated in a wide variety of pathological and physiological processes including signal transduction, cell communication, growth and development, embryogenesis, immune response, hematopoiesis cell survival and differentiation, inflammation, tissue repair and remodeling, atherosclerosis and cancer.
Several gene families are known to encode diverse groups of structurally and functionally related proteins referred to as "growth factors." One such growth factor family includes epidermal growth factor (EGF), transforming growth factor alpha (TGF.alpha.), betacellulin, amphiregulin, and vaccinia growth factor, which are growth and differentiation modulatory proteins produced by a variety of cell types, either under normal physiological conditions or in response to exogenous stimuli. These peptide growth factors influence cells through autocrine and paracrine mechanisms, playing important roles in normal wound healing in tissues such as skin, cornea and the gastrointestinal tract. They also share substantial amino acid sequence homology including the conserved placement of three intrachain disulfide bonds. In addition, all the factors of this EGF-related family bind to a 170,000 molecular weight transmembrane glycoprotein receptor and activate the tyrosine kinase activity in the receptor's cytoplasmic domain (Buhrow, S. A. et al., J. Biol. Chem., 258:7824-7826 (1983)). The receptors are expressed by many types of cells including skin keratinocytes, fibroblasts, vascular endothelial cells, and epithelial cells of the GI tract.
The EGF-related peptide growth factors are synthesized by several cells involved in wound healing including platelets, keratinocytes, and activated macrophages. These growth factors have also been implicated in both the stimulation of growth and differentiation of certain cells, for example, in neoplasia, and the inhibition of other types of cells. For instance, betacellulin is a potent mitogen for retinal pigment epithelial cells and vascular smooth muscle cells. Amphiregulin is a bifunctional cell growth regulatory factor which exhibits potent inhibitory activity on DNA synthesis in neoplastic cells, yet promotes the growth of certain normal cells. A wide variety of uses for amphiregulin have been assigned including the treatment of wounds and cancers. For example, amphiregulin has potent anti-proliferative effects in vitro on several human cancer cell lines of epithelial origin. Amphiregulin also induces the proliferation of human foreskin fibroblasts as shown in U.S. Pat. No. 5,115,096. TGF.alpha. has pleiotropic biological effects and is synthesized by a number of oncogenically transformed, as well as by a variety of tumors, including renal, breast and squamous carcinomas, melanomas and glioblastomas. TGF.alpha. also plays a role in normal embryonic development and adult physiology and is expressed in many tissues including skin, brain, gastrointestinal mucosa and activating macrophages (Derynck, R. Adv. Cancer Res. 58:27-5 (1992)). Accordingly, TGF.alpha. is an important factor in controlling growth of epithelial cells and is important in wound healing, and it has also been found to be angiogenic (Schreiber, et al., Science, 2321250-1253 (1986)).
The transforming growth factor-beta (TGF-.beta.) family of peptide growth factors includes five members, termed TGF-.beta.1 through TGF-.beta.5, all of which form homo-dimers of approximately 25 kDa. The TGF-.beta. family belongs to a larger, extended super family of peptide signaling molecules that includes the Muellerian inhibiting substance (Cate, R. L. et al., Cell, 45:685-698 (1986)), decapentaplegic (Padgett, R. W. et al., Nature 325:81-84 (1987)), bone morphogenic factors (Wozney, J. M. et al., Science 242:1528-1534 (1988)), vg1 (Weeks, D. L., and Melton, D. A., Cell 51:861867 (1987)), activins (Vale, W. et al., Nature, 321:776-779 (1986)), and inhibins (Mason, A. J. et al., Nature 318:659-663 (1985)). These factors are similar to TGF-.beta. in overall structure, but share only approximately 25% amino acid identity with the TGF-.beta. proteins and with each other. All of these molecules are thought to play important roles in modulating growth, development and differentiation. TGF-.beta. was originally described as a factor that induced normal rat kidney fibroblasts to proliferate in soft agar in the presence of epidermal growth factor (Roberts, A. B. et al., Proc. Natl. Acad. Sci. USA 78:5339-5343 (1981)). TGF-.beta. has subsequently been shown to exert a number of different effects in a variety of cells. For example, TGF-.beta. can inhibit the differentiation of certain cells of mesodermal origin (Florini, J. R. et al., J. Biol. Chem. 261:1659-16513 (1986)), induced the differentiation of others (Seyedine, S. M. et al., Proc. Natl. Acad. Sci USA 82:2267-2271 (1985)), and potently inhibit proliferation of various types of epithelial cells, (Tucker, R. F., Science 226:705-707 (1984)). This last activity has lead to the speculation that one important physiologic role for TGF-.beta. is to maintain the repressed growth state of many types of cells. Accordingly, cells that lose the ability to respond to TGF-.beta. are more likely to exhibit uncontrolled growth and to become tumorigenic. Indeed, the cells lack certain tumors such as retinoblastomas lack detectable TGF-.beta. receptors at their cell surface and fail to respond to TGF-.beta., while their normal counterparts express self-surface receptors in their growth is potently inhibited by TGF-.beta. (Kim Chi, A. et al., Science 240:196-198 (1988)). TGF-.beta.1 has been shown to be a multi-functional regulator of cell growth and differentiation (Sporn et al., Science 233:532-534 (1986)) being capable of such diverse effects as inhibiting the growth of several human cancer cell lines, T and B lymphocytes (Kehrl et al., J. Exp. Med. 163:1037-1050 (1986)), inhibition of early hematopoietic progenitor cell proliferation (Goey et al., J. Immunol. 143:877-880 (1989)), stimulating the induction of differentiation of rat muscle mesenchymal cells and subsequent production of cartilage-specific macro molecules (Seyedine et. al., J. Biol. Chem. 262:1946-1949 (1986)), causing increased synthesis and secretion of collagen (Ignotz et al., J. Biol. Chem. 261:4337-4345 (1986)), stimulating bone formation (Noda et al., Endocrinology, 124:2991-2995 (1989)), and accelerating the healing of incision wounds (Mustoe et al., Science 237:1333-1335 (1987)). Further, TGF-.beta.1 stimulates formation of extracellular matrix molecules in the liver and lung. When levels of TGF-.beta.1 are higher than normal, formation of fiber occurs in the extracellular matrix of the liver and lung which can be fatal. High levels of TGF-.beta.1 occur due to chemotherapy and bone marrow transplant as an attempt to treat cancers such as breast cancer. A second protein termed TGF-.beta.2 has been isolated from several sources including demineralized bone, a human prostatic adenocarcinoma cell line (Ikeda et al., Biol. Chem. 26:2406-2410 (1987)). TGF-.beta.2 shares several functional similarities with TGF-.beta.1. These proteins are now known to be members of a family of related growth modulatory proteins including TGF-.beta.3 (Ten-Dijke et al., Proc. Natl. Acad. Sci. USA 85:471-479 (1988)), Muellerian inhibitory substance and the inhibins.
Fibroblast growth factors (FGFs) represent another growing family of peptide growth factors with diverse activities (Miyamoto, M. et al., Mol. and Cell. Biol. 13(7):4251-4259 (1993)). These proteins share the characteristic of binding to heparin and are, therefore, also called heparin binding growth factors (HBGF). Expression of different members of the FGF family is found in various tissues under various temporal and spatial controls. These proteins are potent mitogens for a variety of cells of mesodermal, ectodermal, and endodermal origin, including fibroblasts, corneal and vascular endothelial cells, granulocytes, adrenal cortical cells, chondrocytes, myoblasts, vascular smooth muscle cells, lens epithelial cells, melanocytes, keratinocytes, oligodendrocytes, astrocytes, osteoblasts, and hematopoietic cells.
Each member of the FGF family has activities overlapping with others and also has its unique spectrum of functions (Burgess, W. H. and Maciag, T., Ann. Rev. Biochem. 58:575-606 (1989)). In addition to the ability to stimulate proliferation of vascular endothelial cells, both FGF-1 and 2 are chemotactic for endothelial cells, and FGF-2 has been shown to enable endothelial cells to penetrate the basement membrane. Consistent with these properties, both FGF-1 and 2 have the capacity to stimulate angiogenesis. Another important feature of these growth factors is their ability to promote wound healing. Many other members of the FGF family share similar activities with FGF-1 and 2 such as promoting angiogenesis and wound healing. Several members of the FGF family have been shown to induce mesoderm formation and to modulate differentiation of neuronal cells, adipocytes and skeletal muscle cells. Other than these biological activities in normal tissues, FGF proteins have been implicated in promoting tumorigenesis in carcinomas and sarcomas by promoting tumor vascularization and as transforming proteins when their expression is deregulated cells (Miyamoto, M., et al., Mol. Cell. Biol. 13(7):4251-4259 (1993)).
The formation of new blood vessels, or angiogenesis, is essential for embryonic development, subsequent growth, and tissue repair (Folkman, J. and Klagsbrun, M., Science 235:442-447(1987). Angiogenesis, however, is an essential part of certain pathological conditions such as neoplasia, for example, tumors and gliomas, and abnormal angiogenesis is associated with other diseases such as inflammation, rheumatoid arthritis, psoriasis, and diabetic retinopathy. Both acidic and basic fibroblast growth factor molecules are mitogens for endothelial cells and other cell types. Angiotropin and angiogenin can induce angiogenesis, although their functions are unclear. A highly selective mitogen for vascular endothelial cells is vascular endothelial growth factor or VEGF (Ferrara, N., et al., Endocr. Rev. 13:19-32, (1992)), also known as vascular permeability factor (VPF). Vascular endothelial growth factor is a secreted angiogenic mitogen whose target cell specificity appears to be restricted to vascular endothelial cells.
The murine VEGF gene has been characterized and its expression pattern in embryogenesis has been analyzed. A persistent expression of VEGF was observed in epithelial cells adjacent to fenestrated endothelium, e.g., in choroid plexus and kidney glomeruli. The data was consistent with a role of VEGF as a multifunctional regulator of endothelial cell growth and differentiation Breier, G. et al. Development 114:521-532 (1992)). VEGF is structurally related to the .alpha. and .beta. chains of platelet-derived growth factor (PDGF), a mitogen for mesenchymal cells and placenta growth factor (PLGF), an endothelial cell mitogen. These three proteins belong to the same family and share a conserved motif. Eight cysteine residues contributing to disulfide-bond formation are strictly conserved in these proteins. Alternatively spliced mRNAs have been identified for both VEGF, PLGF and PDGF, and these different splicing products differ in biological activity and in receptor-binding specificity. VEGF and PDGF function as homodimers or heterodimers and bind to receptors which elicit intrinsic tyrosine kinase activity following receptor dimerization.
The temporal and spatial expression of VEGF has been correlated with physiological proliferation of the blood, and its high affinity binding sites are localized only on endothelial cells in tissue sections (Gajdusek, C. M., and Carbon, S. J., Cell Physiol. 139:570-579 (1989)); McNeil, P. L., Muthukrishnan, L., Warder, E., D'Amore, P. A., J. Cell. Biol. 109:811-822 (1989)). The factor can be isolated from pituitary cells and several tumor cell lines, and has been implicated in some human gliomas. Expression of some forms of VEGF confers on Chinese hamster ovary cells the ability to form tumors in nude mice (Ferrara, N., et al., J. Clin. Invest. 91:160-170 (1993)), and inhibition of VEGF function by anti-VEGF monoclonal antibodies was shown to inhibit tumor growth in immune-deficient mice (Kim, K. J., Nature 362:841-844 (1993)). Further, a dominant-negative mutant of the VEGF receptor has been shown to inhibit growth of glioblastomas in mice. Vascular permeability factor has also been found to be responsible for persistent microvascular hyperpermeability to plasma proteins even after the cessation of injury, which is a characteristic feature of normal wound healing. This suggests that VPF is an important factor in wound healing. Brown, L. F. et al., J. Exp. Med. 176:1375-9 (1992). The expression of VEGF is high in vascularized tissues (e.g., lung, heart, placenta and solid tumors) and correlates with angiogenesis both temporally and spatially. VEGF has also been shown to induce angiogenesis in vivo. Since angiogenesis is essential for the repair of normal tissues, especially vascular tissues, VEGF has been proposed for use in promoting vascular tissue repair (e.g., in atherosclerosis).
Thus, there is a need for polypeptides that function as growth factors in regulating a wide variety of developmental and physiological processes, since disturbances of such regulation may be involved in disorders relating to development, hemostasis, angiogenesis, tumor metastisis, cellular migration and ovulation, as well as neurogenesis. Therefore, there is a need for identification and characterization of new families of mammalian growth factors, particularly novel human growth factors, which can play a role in detecting, preventing, ameliorating or correcting such disorders.
Recently, insect homologs of several mammalian growth factors and their receptors have been genetically identified in Drosophila and suggested to play roles in oogenesis (Padgett, R. W. et al., Nature 325:81-84 (1987)); Fergoso, E. L. and Anderson, K. V., Cell 71:451-461 (1992); Stoehling-Hampton, K. et al., Nature 372:783-786 (1994); Panganiban, G. E. et al., Mol. Cell Biol. 10:2669-2677 (1990)), embryogenesis (Xia, T. et al., Science 283: 1756-1759 (1994); Raz, E. and Shilo, B. Z., Genes & Dev. 7: 1937-1943 (1993); Brand, A. H. and Perriman, N., Genes & Dev. 8: 629-639 (1994); Goode, S. et al., Development 116:177-192 (1992); Livneh, E. et al., Cell 40:599-607 (1985); Neuman-Silverberg, F. S. and Schupbach T., Cell 75:165-174 (1993)) and morphogenesis of specified organs (Heberlain, U. et al., Cell 75:913-926 (1993); Nellen, D. et al., Cell 78:225-237 (1994); Brummel, T. J. et al., Cell 78:251-261 (1994); Penton, A. et al., Cell 78:239-250 (1994)). For instance, a member of the Drosophila TGF-.beta. family, decapentaplegic (dpp), was shown to act as a morphogen for dorsal-ventral pattern organization in Drosophila (Fergoso, E. L. and Anderson, K. V., supra).
However, a prototype for a new polypeptide growth factor family, for which no mammalian homolog was known, has recently been identified in insects (K. Homma et al., J. Biochem. 271:13370-13775 (1996)). This growth factor, termed "Insect-Derived Growth Factor (IDGF)," was isolated from the conditioned medium of NIH Sape-4 cells, an embryonic cell line of the flesh fly, and was purified to homogeneity. Like many other cell lines established from various insects, NIH Sape-4 cells inoculated at high density proliferated in the absence of fetal calf serum and known growth factors. As suggested by this finding, these embryonic insect cells were found to produce a growth factor, IDGF, that stimulates their proliferation in an autocrine manner. IDGF is a homodimer of a protein with a molecular mass of 52 kDa, and its specific activity for stimulating replication of cultured embryonic cells is comparable to those of mammalian growth factors in stimulating target cell replication. Imunoblotting experiments revealed that unfertilized mature eggs of the flesh fly contained this growth factor, a certain level of which was maintained throughout embryonic development. Analysis of cDNA for the growth factor showed that this factor is a novel protein consisting of 553 amino acids. No significant sequence similarity was found between this factor and other proteins except 25% amino acid identity shared with atrial gland granule-specific antigen (AGSA) of Aplysia californica, suggesting that this insect antigen for which no function was previously known is also a growth factor.
It is also known that dendritic cells (DC) are the principal antigen presenting cells involved in primary immune responses; their major function is to obtain antigen in tissues, migrate to lymphoid organs, and activate T cells (Mohamadzadeh, M. et al., J. Immunol. 156: 3102-3106 (1996). For example, Langerhans cells (LC), which are skin-specific members of this family, have been shown to present a variety of antigens that may be generated in or penetrate into skin. In contact hypersensitivity, topical application of a reactive hapten activates LC to migrate out of the epidermis into draining lymph nodes, where they present this antigen to selected T cells. Human LC lines secrete relatively large amounts of various chemokines such as NAP-1/IL-8 and MIP-1.alpha. upon ligation of CD40 on cell surfaces. Thus, it is likely that LC possess the potential to produce a selected set of chemokines with chemotactic activities for T cells.
DC are also the first immune cells to arrive at sites of inflammation on mucous membranes, the major site of sexual transmission of HIV. Weissman, D. et al., J. Immunol. 155:4111-4117 (1995) Mature DC in peripheral blood bind HIV to their surface and induce infection when added to autologous CD4+ T cells in the absence of added stimuli, such as mitogens. These mature DC, when isolated directly from peripheral blood, appear to be conjugated to T cells, and these conjugates are infected easily and productively with HIV. These findings suggest a role for DC in early HIV infection in which they bind virus and interact with T cells locally or after migrating to a lymphoid organ, thus establishing a productive infection. Furthermore, they likely play a role in the propagation of HIV infection by activating T cells in the presence of HIV, which leads to viral replication and immune cell destruction. Thus, there is a need for identifying new polypeptide factors which may mediate interactions between DC and T cells which may lead to T cell activation or HIV infection.
DiGeorge Syndrome, also called thymic aplasia (failure of organ to develop naturally), thymic hypoplasia (defective development of tissue), or third and fourth pharyngeal arch or pouch syndrome, is a congenital immune disorder characterized by lack of embryonic (stage in prenatal development between 2-8 weeks inclusive) development or underdevelopment of the pharyngeal pouches. The syndrome is often associated with congenital heart defects, anomalies of the great vessels, esophageal atresia (congenital failure of esophageal tube to develop) and abnormalities of facial structures, and low levels of serum calcium as a result of the hypoparathyroidism (insufficient secretion of the parathyroid glands). In most cases there is an observable chromosomal defect on chromosome 22.
Pathologically, DiGeorge Syndrome is characterized by absence or hypoplasia of the thymus and parathyroids with varying degrees of T cell immunodeficiency. Deficiency of cell-mediated immune response may result in increased susceptibility to infection. Depending on the degree of parathyroid or thymic hypoplasia, hypocalcemic tetany (intermittent, tonic spasms, paroxysmal and involving the extremities) may be present. DiGeorge Syndrome arises from a disturbance of normal embryologic development of the pharyngeal pouches between the sixth and 10th weeks of gestation. This disturbance can affect the first, second, third, fourth and sixth pharyngeal pouches, depending on when during this key time period the disturbance occurs. For example, a disturbance of the third and fourth pharyngeal pouches affects development of the thymus and aorta. Disturbances in the third pharyngeal pouch also affect development of the parathyroid, while disturbances in the first and second pharyngeal pouches affect lip and external ear development. Development of the pulmonary artery is influenced by the sixth pharyngeal pouch. Thus, to aid in the diagnosis and treatment of DiGeorge syndrome and similar pathologies, there is a need to identify polypeptide factors which map near the chromosome 22 locus associated with DiGeorge syndrome, are expressed early in development, and also have effects on embryonal cells.